Our ability to hear is made possible by way of a Rube Goldberg-style
process in which sound vibrations entering the ear shake and jostle
a successive chain of structures until, lo and behold, they are
converted into electrical signals that can be interpreted by the
brain. Exactly how the electrical signal is generated has been
the subject of ongoing research interest.

In a study published in the September 6 issue of the journal Nature,
researchers have shed new light on the hearing process by identifying
two key proteins that join together at the precise location where
energy of motion is turned into electrical impulses. The discovery,
described by some scientists as one of the holy grails of the field,
was made by researchers at the National Institute on Deafness and
Other Communication Disorders (NIDCD), one of the National Institutes
of Health (NIH), and the Scripps Research Institute in La Jolla,
CA.

"This team has helped solve one of the lingering mysteries
of the field," says James F. Battey, Jr., M.D., Ph.D., director
of the NIDCD. "The better we understand the pivotal point
at which a person is able to discern sound, the closer we are to
developing more precise therapies for treating people with hearing
loss, a condition that affects roughly 32.5 million people in the
United States alone."

When a noise occurs, such as a car honking or a person laughing,
sound vibrations entering the ear first bounce against the eardrum,
causing it to vibrate. This, in turn, causes three bones in the
middle ear to vibrate, amplifying the sound. Vibrations from the
middle ear set fluid in the inner ear, or cochlea, into motion
and a traveling wave to form along a membrane running down its
length. Sensory cells (called hair cells) sitting atop the membrane "ride
the wave" and in doing so, bump up against an overlying membrane.
When this happens, bristly structures protruding from their tops
(called stereocilia) deflect, or tilt to one side. The tilting
of the stereocilia cause pore-sized channels to open up, ions to
rush in, and an electrical signal to be generated that travels
to the brain, a process called mechanoelectrical transduction.

Most scientists believe that the channel gates are opened and
closed by microscopic bridges — called "tip links" — that
connect shorter stereocilia to taller ones positioned behind them.
If scientists could determine what the tip links are made of, they'd
be one step closer to understanding what causes the channel gates
to open. This is no easy feat, however, because stereocilia are
extremely small, scarce, and difficult to handle. Several proteins
had been reported to occur at the tip link in earlier studies,
but results have been conflicting to this point.

Cadherin 23 and Protocadherin 15 Unite to Form Tip Link

Using three lines of evidence, NIDCD scientists Hirofumi Sakaguchi,
M.D., Ph.D., Joshua Tokita, and Bechara Kachar, M.D., together
with Piotr Kazmierczak and Ulrich Müller, Ph.D., of Scripps Research
Institute, and other collaborators have demonstrated that two proteins
associated with hearing loss — cadherin 23 (CDH23) and protocadherin
15 (PCDH15) — unite and adhere to one another to form the
tip link. Mutations in CDH23 are known to cause one form of Usher
syndrome as well as a nonsyndromic recessive form of deafness,
and mutations in PCDH15 are responsible for another form of Usher
syndrome. (A syndrome is a disease or disorder that has more than
one feature or symptom, while the term "nonsyndromic" refers
to a disease or disorder that is not associated with other inherited
characteristics.) Usher syndrome is the most common cause of deaf-blindness
in humans.

"Cadherin 23 and protocadherin 15 have been implicated in
a variety of forms of late- and early-onset deafness, and a whole
range of mutations can produce different outcomes," says NIDCD’s
Kachar, a co-senior investigator on the study. "Now that we
know how these two proteins interact at the tip link, we can perhaps
predict how different types of hearing loss can take place depending
on where a mutation is located."

Three Lines of Evidence

The researchers first created antibodies that would bind to and
label short segments on the CDH23 and PCDH15 proteins in the inner
ears of rats and guinea pigs. (Both proteins were identified at
the tip link, respectively, in earlier studies.) Using green fluorescence
and electron microscopy studies, they showed that CDH23 was located
on the side of the taller stereocilium and PCDH15 was present on
the tip of the shorter one, with their loose ends overlapping in
between. The researchers were able to identify both proteins, while
earlier studies had not, because they removed an obstacle to the
antibody-binding process: calcium. Under normal conditions, CDH23
and PCDH15 are studded with calcium ions, which prevent antibodies
from binding to the targeted sites. When calcium was removed through
the addition of a chemical known as BAPTA, both labels became visible.

Next, the researchers built a structure resembling a tip link
by expressing the CDH23 and PCDH15 proteins in the laboratory and
watching how they interacted. When conditions were right, the two
proteins wound themselves tightly together from one end to the
other in a configuration that mirrored a naturally occurring tip
link. The results were surprising, since the scientific consensus
had been that these proteins would not interact at all. As with
normal tip links, the structure thrived in calcium concentrations
that paralleled those found in fluid of the inner ear, while a
drastic reduction in calcium disrupted the structure.

Lastly, the scientists found that one mutation of PCDH15 that
causes one form of deafness inhibited the interaction of the two
proteins, leading them to conclude that the mutation reduces the
adhesive properties of the two proteins and prevents the formation
of the tip link. In a second mutation of PCDH15, the tip link was
not destroyed; the scientists suggested that the deafness is not
likely caused by the breakup of the tip link but by interference
with its mechanical properties.

Knowing precisely the composition and configuration of the tip
link, scientists can now explore how these proteins interact with
other components to form the rest of the transduction machinery.
In addition, scientists can study how new treatments might be developed
to address the breaking up of tip links through environmental factors,
such as loud noise.

"Now that we understand what the tip link is made of and
what conditions are required to assemble it," says Kachar, "we
can study what it might take to rejoin tip links as a possible
method for restoring hearing in people with some forms of hearing
loss that may have resulted from disruption of the tip link."

Funding of the study was principally provided by the NIDCD. Other
NIH institutes and centers that contributed funding were the National
Institute of General Medical Sciences (NIGMS), the National Institute
of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), and
the National Center for Research Resources (NCRR).

The NIDCD supports and conducts research and research training
on the normal and disordered processes of hearing, balance, smell,
taste, voice, speech, and language and provides health information,
based upon scientific discovery, to the public. For more information
about NIDCD programs, see the Web site at www.nidcd.nih.gov.

The National Institutes of Health (NIH) — The Nation's
Medical Research Agency — includes 27 Institutes and
Centers and is a component of the U.S. Department of Health and
Human Services. It is the primary federal agency for conducting
and supporting basic, clinical and translational medical research,
and it investigates the causes, treatments, and cures for both
common and rare diseases. For more information about NIH and
its programs, visit www.nih.gov.